This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Progressive obstructive lung disease is the leading cause of morbidity and mortality in CF. Pulmonary infections are common in CF and the majority of patients will become chronically colonized with Pseudomonas aeruginosa despite an intact immune system and a vigorous immune response. Early colonization and conversion of the P. aeruginosa to its mucoid form are associated with more rapid loss of lung function and increased mortality. Once CF patients develop chronic colonization with P. aeruginosa they are often unable to clear the infection even with use of intensive antibiotics. P. aeruginosa has developed ways to escape both the innate and adaptive immune response by forming biofilms and releasing a variety of virulence factors. However, the nature of the CD4 T cell adaptive immune response generated to P. aeruginosa may dramatically alter the balance between clearance of the pathogen and induction of tissue damage. While Th1 (IFNg) responses are important for bacterial clearance through macrophage and neutrophil activation, Th2 (IL-4) responses primarily result in antibody production, particularly IgE, and are not effective in the clearance of bacteria. In mouse models of chronic P. aeruginosa infection, mice with a Th1 immune response had better bacterial clearance, milder lung inflammation and improved survival compared to mice with Th2 immune responses and IFNg (Th1) was shown to be an effective treatment of airway inflammation in a rat model of chronic P. aeruginosa lung infection. Total peripheral blood mononuclear cells (PBMCs) from CF patients chronically colonized with P. aeruginosa produced higher levels of IL-4 (Th2) and lower levels of IFNg (Th1) in response to Pseudomonal antigens than PBMCs from uninfected CF patients. Whole blood cultures from CF children chronically infected with P. aeruginosa had greater IL-4 production than did cultures from noninfected CF patients. Additionally, a recent study showed that CF patients chronically colonized with P. aeruginosa had higher levels of Th2 cells and IL-4 and lower levels of IFNg in bronchial alveolar lavage (BAL) when compared to noninfected CF patients and healthy controls. Additionally, Th2 bias to P. aeruginosa has also been associated with lower lung function in CF patients raising the possibility that shifting immune responses toward a Th1 phenotype, and away from a Th2 phenotype could potentially lead to improved outcomes in chronically P. aeruginosa-infected patients. CF patients also have a high prevalence of asthma and ABPA, both of which are diseases that cause damaging airway inflammation and are mediated by activation of Th2 lymphocytes. The predominance of Th2 immune responses in CF patients is further supported by two studies showing that CF patients, compared to healthy controls, had increased levels of serum and sputum cationic protein, an indicator of Th2 cell induced eosinophilic activation. These data suggest that not only is a Th2 immune response associated with ineffective airway clearance of P. aeruginosa, it may also contribute to damaging airway inflammation and a more rapid lung function decline. Modulation of Th2 immune responses in CF may provide new therapeutic targets especially in patients chronically colonized with P. aeruginosa. Mutations of CFTR in epithelial cells do not readily explain the exaggerated inflammation characteristic of CF lung disease. CD4 T cells are important in the generation of an adaptive immune response, can regulate chronic inflammation and are present in large numbers in the subepithelial space surrounding the CF airway. CD4 T cells express CFTR and therefore may be affected by mutations in this gene. CD4 T cells can differentiate into several effector subtypes including Th1 cells that make IFNg and Th2 cells that make IL-4. How CFTR mutations affect CD4 T cell function is poorly understood. Our lab has recently shown that activated naive CD4 T cells from CF mice produce greater amounts of IL-4 (Th2) than wild type (WT) mice while IFNg (Th1) production is equivalent, suggesting that CFTR is regulating CD4 T cell differentiation. We have also shown that intracellular calcium levels, an important signal generated by T cell receptor activation, are increased in activated CD4 T cells from CF mice when compared to WT and that there is increased nuclear binding of the IL-4 transcription factor, NFAT. Calcium is required for NFAT cytoplasmic to nuclear translocation by activation of calcineurin which in turn dephosphorylates NFAT. We have confirmed the in vivo significance of these findings by showing that CF mice, when compared to WT mice, generate an exaggerated Th2 immune response following exposure to A. fumigatus and have increased CD4 T cell IL-4 production (Th2) following A. fumigatus and P. aeruginosa exposure. These results implicate a primary CD4 T cell immune defect in CF and strongly suggest that CFTR regulates CD4 T cell differentiation (Th1 versus Th2). Further, these findings have significant clinical implications given the growing data that Th2 immune responses are associated with chronic P. aeruginosa colonization and poor lung function. There is growing evidence that there is a Th2 biased immune response in CF and that this bias is associated with ineffective clearance of P. aeruginosa infection. While much focus has been on CFTR function in epithelial cells, we propose a novel mechanism for the role of CFTR in CD4 T cells. In a CF mouse model we have shown that CFTR may regulate CD4 T cell differentiation such that mutations favor a Th2 bias.